Deubiquitinating enzymes (DUBs, also known as deconjugating enzymes) are important regulatory proteases that process the primary gene products of the ubiquitin gene family, reverse the conjugation of ubiquitin to cellular proteins, and disassemble the polyubiquitin chains that target proteins for degradation by the proteasome. As such, they regulate proteolysis and thereby progression through the cell cycle, signal transduction pathways, the stress response, antigen presentation, chromatin structure, receptor internalization and endocytosis. It has only recently been appreciated that modification of proteins by other ubiquitin-like (Ubl) proteins exerts a similar targeting function. The underlying assumption of this grant is that regulation of the Ubl pathways by deconjugation of ubiquitin-like proteins is likely to be equally important, with roles in cancer, the regulation of growth and apoptosis. The enzymes that reverse the conjugation of UbI proteins (Ubiquitin-like protein proteases, ULP) are attractive targets for further study since the reversal of these modifications is expected to interfere with functions. As they are proteases, a large body of work suggests approaches to effective pharmacological intervention. Additionally, it is easier (conceptually and practically) to interfere with these pathways by overexpressing a deconjugating enzyme than it is to prevent attachment of the UbI domain; an approach that requires gene knock-out or dominant negative approaches. Understanding the role of these deconjugating enzymes requires that we know more about this class of enzymes, their substrates and their regulation. A series of inhibitors and substrates for ULPs will be synthesized and used to test three specific hypotheses. First, Dr. Wilkinson will test the hypothesis that an integral subunit of the mammalian COP9-signalosome is able to reverse the Nedd8 modification of cullins. He also will test the hypothesis that modification of proteins by ISG15 are an integral part of the interferon response by identifying, and interfering with the function of, enzymes which reverse this modification. He will determine if conjugation of the proapoptotic Ubl FAT10 is reversible. Finally, he will test the hypothesis that HAUSP and/or SENPI are the Ubl that removes SUMO-1 from the Promyelocytic leukemia protein, triggering the disassembly of PML.